Gene delivery is a promising method for the treatment of acquired and inherited diseases. Gene delivery to the nervous system presents several problems including the quiescent nature of neuronal cells and the blood-brain barrier for delivery of genes to the brain. A majority of gene therapy trials have used retroviral-mediated gene transfer, which typically is used for gene delivery into dividing somatic cell populations and requires cell division for integration and expression. The requirement for cell division in retroviral gene therapy makes the use of retroviruses for gene delivery to the nervous system unlikely.
More recently, other types of viral vectors have been used, including recombinant vectors based on AAV particles. The viral genome of AAV consists of a 4.7 kb single-stranded DNA molecule, which is composed of two 145-base inverted terminal repeats (ITRs) flanking two open reading frames, Rep and Cap. In vectors designed for gene delivery, the Rep and Cap open reading frames are deleted and the only viral sequences in the rAAV vectors are the ITRs. AAV is a helper-dependent DNA parvovirus which belongs to the genus Dependovirus. AAV has a wide host range and is able to replicate in cells from any species so long as there is also a successful infection of such cells with a suitable helper virus. AAV has not been associated with any human or animal disease. For a review of AAV, see, e.g., Berns and Bohenzky, Advances in Virus Research (Academic Press, Inc.) 32:243-307, 1987.
Recombinant adeno-associated viral (rAAV) vectors have several properties that make them one of the most promising vehicles for gene delivery to the central nervous system (CNS). rAAV vectors have been reported to infect and transduce both dividing and non-dividing cells, including neurons with minimal cellular toxicity or host immune response (Peel et al., J. Neurosci. Methods, 98:95-104, 2000). In the central nervous system (CNS), significant long-term transduction of neurons by rAAV has been observed for up to 1 year (Xu et al., Gene Therapy, 5: 1323-1332, 2001; Lo et al., Hum. Gene Ther., 10: 201-213, 1999).
Previously, use of rAAV was hampered by the lack of methods for producing high titer vectors that were not contaminated with helper viruses, such as adenovirus or herpes simplex virus. Recent developments in helper virus-free packing systems and a new purification protocol have made possible the production of large-scale high titer rAAV free of contaminating helper virus (Grimm et al., Hum. Gene Ther., 9: 2745-2760, 1998; Xiao et al., J. Virol., 72: 2224-2232, 1998; Clark et al., Hum. Gene Ther., 10: 1031-1039, 1999; Zolotukhin et al., Gene Therapy 6: 973-985, 1999).
An important consideration in applying gene delivery to the CNS is the effect on the patient that may result from chronic, continuous expression in the nervous system of a biologically active molecule that could affect cells in addition to the target cells. This consideration is particularly relevant for neurotrophic factors since these are secreted molecules whose receptors are often widespread in the CNS. Therefore, an ideal vector for in vivo gene therapy of a nervous system disorder should include not only the ability to effectively and safely transduce the therapeutic gene into the nervous system, but also the ability to temporally regulate gene expression.
Regulatable promoter systems have been studied for transgene regulation in mammalian cells. Examples of promoter systems that have been developed for regulatable gene expression systems include a tetracycline-responsive (tet), a RU-486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter. Previously, gene delivery vectors have been used for delivery of therapeutic genes without regulatable elements.
The tet response element-based system is well characterized and was first described by Gossen and Bujuard (Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992). The tet system includes several advantages. The elements required for control of the tet system are derived from a prokaryotic organism, and thus, there is no endogenous expression of these control elements in mammalian cells. In addition, a preferred effector doxycycline (dox), a tet derivative, is an FDA approved drug that can regulate the transgene expression at very low concentrations without producing detectable side effects (Corti et al., Nat. Biotechnol, 17: 349-354, 1999; Hasan et al., Genesis, 29: 116-122, 2001). Activation of the tet-regulated system by dox is dose-dependent and gene expression can be controlled over a narrow window of dox concentrations (Urlinger et al., Proc. Natl. Acad. Sci. USA, 97: 7963-7969, 2000). The genes required for the tet system are small compared to elements required for other systems, which is advantageous given the limited insert size of approximately 4.5 kb, for rAAV vectors (Baron et al., Methods Enzymol., 327: 401-421, 2000).
The tet inducible system includes two components, the tet-controlled transactivator protein (tTA), and the tet-regulated element (TRE). The tTA is a fusion protein of the tet repressor DNA binding domain of Escherichia coli (TetR) and the C-terminal transcriptional activator domain of the VP16 protein from herpes simplex virus. The TRE region includes seven copies of the tetracycline resistance operator binding sites and a minimal cytomegalovirus (CMV) promoter region that contains the TATA box and transcription start sites. In the absence of tet or dox, tTA can bind a tetracycline operator (tetO) sequence located in front of the minimal promoter and stimulate transcription of the transgene. Dox prevents this binding and consequently abolishes transcription because the minimal promoter by itself is inactive. This tet-off system has also been modified to make a tet-on system. When the tTA is replaced by a mutated transactivator, rtTA, the promoter regulation by the transactivator is reversed so that transgene expression occurs in the presence of dox and is shut off in the absence of dox. (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992; Urlinger et al., Proc. Natl. Acad. Sci. USA, 97: 7963-7969, 2000; Baron et al., Methods Enzymol., 327: 401-421, 2000).
The tet-off and tet-on systems have been studied in the context of various viral vectors. In some cases, the two components of the tet system have been cloned into separate viruses, which requires coinfection of the target cells by both viruses to obtain regulatable transgene expression (see, for example, Bohl et al., Hum. Gene Ther., 8:195-204, 1997). Another strategy is to combine both components into one self-regulating virus so that target cells only need to be infected by one virus to allow regulatable expression. (see, for example, Corti et al., Nat. Biotechnol., 17: 349-354, 1999).
However, the limited insert size of in rAAV for foreign genes makes it difficult to design a tet-regulatable vector with an insulator region between the two tet expression cassettes to minimize promoter activity originating from the ITRs which are necessary for rAAV packaging (Fitzsimons et al., Gene Therapy, 8: 1675-1681, 2001; Flotte et al., J. Biol. Chem., 268: 3781-3790, 1993). This limitation may result in leaky regulation of gene expression in the context of vector backbone.
A need exists for a tightly regulatable gene delivery vector for delivery of therapeutic genes to the nervous system.